Despite the huge advances of molecular biology, there are still only primitive tools to describe how biomolecules do their work. Our Section on Molecular Biophysics measures, characterizes and codifies the forces that govern the organization of all kinds of biological molecules. Our undertaking is strengthened by its strong connection with physical theory. Our long-term goal is to build a practical physics of biological material. We aim to realize this goal through a series of measurements and analyses of the different kinds of forces as revealed in vivo, in vitro and in computation. In particular, we are working with DNA/lipid assemblies for gene therapy; DNA assemblies such as are seen in viral capsids and in vitro; polypeptides and polysaccharides in suspension; and lipid/water liquid-crystals. In all these systems, we simultaneously observe the structure of packing as well as measure intermolecular forces or interaction energies. Because ions vary widely in their effects on biological materials, ion "specificity" beyond simple charge properties is a major issue in biology. One overlooked property of ions is the polarizability, the ability of the charge to shift or fluctuate, a property seen in charge fluctuation forces. Another surprising feature of ions is the tendency to stick to charged bilayers, stick to an extent beyond what is expected from charge-charge attraction. This stickiness changes the way membranes interact, it also introduces strains that can alter the the way proteins are accommodated and are able to change conformation as in the opening and closing of trans-membrane ionic channels. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties. Using X-ray diffraction and NMR spectroscopy, we found that in the presence of sodium ions negatively charged phosphatidylserine (PS) bilayers have lateral areas much smaller than the areas of corresponding neutral phosphatidylcholines (PC) analogs. This shrinkage occurs despite the extra electrostatic repulsion expected for charged PS headgroups. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. We find that the charged bilayers repel as expected when interlamellar forces are measured by osmotic stress and x-ray diffraction. Why does this same repulsion not occur between charged groups on the same bilayer? Why are the charged lipids stiffer against bending? The culprit is likely strong ion binding. Indeed we found a few years ago that protons bind so strongly to these same lipids that at 10-4 Molar concentrations (pH 4), the membrane is wrenched from the lamellar form into an inverted hexagonal structure. More intriguing, when these same lipids are used as a scaffold for protein channels, exposure to protons shifts channel gating. Suddenly we have become aware of the organizing power of small ions changing membrane protein behavior. Guided by the unexpectedly strong attraction between charged phospholipids and simple ions, we have begun to examine Li ions with phosphatidylserine bilayers. Why? Earlier work by others suggests that Li acts like protons. We are now asking whether Li can stress lipids assembly and consequently whether low levels of Li modify channel gating. Structural studies have progressed during the past year, including work at CHESS (Cornell High Energy Synchrotron Source) where we obtain high-resolution data needed for measurement of fluctuations, forces and stress in protein-hosting lipids. Weak compared to covalent bonds and to electrical-charge interactions, charge-fluctuation or van der Waals forces are not usually appreciated for the work they do among membranes and macromolecules. The dominant force that coheres membranes and proteins, source of the powerful surface tension at membrane interfaces, van der Waals forces are again the dominant -- perhaps sole -- attraction that creates membrane multilayers or allows membranes to adhere to artificial surfaces. This past year has been a festival of learning to formulate, to measure, and to modify these neglected interactions. The key has been to begin with the elements of physical theory and to relate the polarizability of materials to the fluctuations of charges within them. From this we have been able to design experiments that show how macromolecular organization responds to deliberate changes in solution properties. We have been able to see how adding even the simplest salts to water around lipid bilayers will change how these membranes assemble into multilayers. For example, we have seen how the attraction between membranes varies when salts of different ions chloride vs. bromide are dissolved in the intervening water. Membrane multilayers will swell by 50% with bromide but not with chloride. We have reformulated van der Waals forces between membranes to show how they would respond to changes in solutions so as to have a strategy to control membrane assembly. One unexpected by-product has been a collaboration with engineers using our equations to design production procedures for thin-film resistors in computer chips. We expect the collaboration to work to our benefit by providing us with experimental data that are used to compute van der Waals forces. Another unexpected result relates to what was known about the friction between an ion and the water through which it moves. Just as there is a charge-water interaction that creates a drag, there is a charge-fluctuation source of drag on a moving particle. Computation: Driven by the conditions set by smaller solutes, proteins "fold" and "unfold." Experimentally, these conditions are stated as intensive variables -- pH and other chemical potentials -- as though small solutes were infinite resources that come at an independently varied price. Computationally, the finite spaces of simulation allow only fixed small numbers of these solutes. By combining the analytic Gibbs adsorption isotherm with the computational Monte Carlo sampling of polymer configurations, we have been able to overcome an inherent limitation of computer simulation. The trick is to compute analytically the free energy changes wrought by solutes on each particular configuration. Then numerical computation is needed only to sample the set of configurations as efficiently as though no bathing solute were present. The result is a more accurate computation procedure five hundred times faster than earlier simulations that had to count all possible positions of the cosolutes in the bathing solution. Experiment: To monitor at how small adherent molecules affect molecular association, we measured the changes of binding free energy vs. change in water activity for the specific binding of cyclodextrin with an adamantane derivative. The dependence of the binding constant on osmotic pressure, using different salts and neutral agents, suggests a release of 15-25 water molecules from the interacting surfaces upon association, depending on the type of solute used. The observed dependence of binding free energy and enthalpy with added solute indicates that these osmolytes are interacting primarily enthalpically with these surfaces.